Dengue viruses (DVs) are positive sense, single-stranded RNA viruses belonging to the Flavivirus genus of the Flaviviridae family. The genomic organization consists of the following elements: 5′ noncoding region (NCR), structural proteins (capsid (C), premembrane/membrane (prM/M), envelope (E)) and nonstructural proteins (NS1-NS2A-NS2B-N53-NS4A-NS4B-NS5), and 3′ NCR. Similar to other flaviviruses, the DV viral genome encodes an uninterrupted coding region which is translated into a single polyprotein. The genomic RNA contains a type I cap at the 5′ end but lacks a poly-A tail at the 3′ end.
The viruses are maintained in a cycle which involves humans and Aedes aegypti as well as Aedes albopictus mosquitoes. The infection in humans is initiated by injection of the virus while an infected mosquito takes a blood meal. The virus in the mosquito's saliva is deposited mainly in the extravascular tissues. The first category of cells infected after inoculation are dendritic cells, which then migrate to the lymph nodes (Wu et al. Nature Med. 7:816-820 (2000)). After an initial replication in the skin and in the lymph nodes, the virus appears in the blood during the acute febrile phase, generally for 3 to 5 days.
Dengue diseases are caused by four virus serotypes (DV1, DV2, DV3, and DV4) of the Flavivirus genus. Infection with a dengue serotype can produce a clinical disease spectrum ranging from a nonspecific viral syndrome to a severe hemorrhagic disease which can be fatal. The incubation period of dengue fever after a mosquito bite is approximately 4 days (ranging from 3 to 14 days). Dengue fever is characterized by a biphasic fever, headaches, pain in various parts of the body, prostration, eruptions, lymphadenopathy and leukopenia (Kautner et al. J. of Pediatrics 131:516-524 (1997); Rigau-Perez et al. Lancet; 352: 971-977 (1998)). The viremia period is the same as for febrile diseases (Vaughn et al. J. Infect. Dis. 176:322-30 (1997)). Recovery from dengue fever occurs after 7 to 10 days, but there is usually a prolonged asthenia. Decreases in leukocyte and platelet count are common.
Hemorrhagic dengue is a severe febrile disease characterized by anomalies in homeostasis and an increase in vascular permeability that can result in hypovolemia and in hypotension (dengue with shock syndrome) often complicated by severe internal hemorrhaging. The mortality rate of hemorrhagic dengue can be up to 10% without treatment, but is 1% in most centers with experience in treatment (WHO Technical Guide, 1986. “Dengue haemorrhagic fever: diagnosis, treatment and control” pp. 1-2. World Health Organization, Geneva, Switzerland).
Dengue is the second most common tropical infectious disease after malaria and more than half the world's population (2.5 billion) lives in regions where there is a risk of epidemic transmission. Each year, cases of dengue are estimated at 50-100 million, cases of patients hospitalized for hemorrhagic dengue at 500 000, and the number of deaths at 25 000. Dengue is endemic in Asia, in the Pacific region, in Africa, in Latin America and in the Caribbean. More than 100 tropical countries are endemic for dengue virus infections and hemorrhagic dengue has been documented in 60 of these countries (Gubler, TRENDS in Microbiology 10:100-103 (2002); Monath. Proc. Natl. Acad. Sci. USA 91:2395-2400 (1994)).
There is no specific therapeutic for diseases caused by infection by dengue virus. The treatment for dengue fever is symptomatic, with confinement to bed, control of fever and pain with antipyretics and analgesics, and adequate fluid intake. The treatment for hemorrhagic dengue requires equilibration of fluid losses, replacement of clotting factors and heparin infusion.
Preventive measures are currently based on controlling the vector and taking personal protection steps which are difficult to implement and expensive. No vaccine against dengue has been approved at this time. Given that the four dengue serotypes are in circulation in the world and since they have been reported as being involved in cases of dengue hemorrhagic fever, immunization should ideally confer protection against the four serotypes of the dengue virus.
Recombinant DNA technology has made it possible to develop live attenuated dengue virus vaccine candidates. Methods have been developed to recover infectious dengue virus from cells transfected with RNA transcripts derived from a full-length cDNA clone of the dengue virus genome, thus making it possible to derive infectious viruses into which attenuating mutations can be introduced (e.g., via the cDNA clone by site-directed mutagenesis).
Thus, the present invention provides methods and compositions for a DV3 infectious clone for use in therapeutic, vaccine and diagnostic applications.